Increased activity of the PI3K/AKT/mTOR pathway has been observed in chronic myeloid leukemia (CML). Morin, a kind of flavonoid, exhibits a significant anticancer activity by suppressing the PI3K/AKT signaling pathway. However, the effect of morin on CML and its underlying mechanisms is poorly understood. Here, we found that morin dose dependently inhibited the proliferation of CML cell lines K562 and KCL22 and induced their apoptosis, with a significant increase in cell apoptosis upon exposure of cells to 50 μmol/L morin. Moreover, morin significantly reduced CML xenograft growth in nude mice. Mechanically, morin attenuated phosphorylated AKT level by upregulating PTEN expression, thus leading to the inhibition of AKT signaling. Knockdown of PTEN by its siRNA completely abrogated morin-induced cell apoptosis, indicating that PTEN mediates the inductive effect of morin on CML cell apoptosis. More importantly, we found that miR-188-5p was significantly upregulated in CML patients and CML cell lines. Treating CML cells with morin markedly downregulated the miR-188-5p expression level. Further, we demonstrated that miR-188-5p repressed PTEN expression by directly targeting its 3′-UTR. miR-188-5p downregulation induced by morin enhanced CML cell apoptosis by relieving miR-188-5p repression of PTEN expression. In summary, morin exerts significant anticancer efficacy in CML by regulating the miR-188-5p/PTEN axis and thus repressing the PI3K/AKT signaling.

Chronic myeloid leukemia (CML) is a gene-driven myeloproliferative disorder that is characterized by the presence of the Philadelphia (Ph) chromosome (1). The BCR–ABL fusion gene, resulting from the reciprocal translocation between chromosomes 9 and 22, leads to the expression of the BCR–ABL tyrosine kinase, which plays a key role in the pathogenesis of CML by activating multiple mitogenic signaling pathways (2). For example, in hematopoietic cells, the level of the cyclin-dependent kinase inhibitor p27Kip1 is regulated by BCR–ABL tyrosine kinase through activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, leading to accelerated entry into the S phase; BCR–ABL tyrosine kinase also mediates the transformation of human hematopoietic cells by Grb2-mediated activation of MAPK (3, 4). In cells transformed by v-ABL, BCR–ABL activates signal transducers and activators of transcription (STAT) family and the downstream proteins of the JAK/STAT pathway (5). Moreover, several antiapoptotic proteins including BCL-2, MCL-1, and XIAP are overexpression in BCR–ABL-expressing cells contributing to the activation of above these signaling pathways, resulting cell survival advantage (6, 7).

Emerging evidence suggests that inhibition of the PI3K/AKT/mTOR pathway activation may have antiproliferative and proapoptotic effects on leukemia (8). The clinical use of imatinib (IM), the first BCR–ABL tyrosine kinase inhibitor, has significantly improved the prognosis of patients with CML compared with previous treatment. However, the resistance and intolerance of IM limit the complete eradication of patients with CML and affect the prognosis of these patients (9). It has been demonstrated that phosphatase and tension homology deleted on chromosome 10 (PTEN) acts as a tumor suppressor by negatively regulating the PI3K/AKT signaling pathway (10). Unfortunately, previous studies have confirmed that the PTEN expression level was downregulated in CML cells, and a reduction of the PTEN level was strongly associated with BCR–ABL expression (11, 12). Additionally, deregulation of PTEN protein expression by monoubiquitination and noncoding RNAs has been known to occupy an important role in its tumor-suppressive effects (13, 14). However, despite the essential importance of PTEN in regulating PI3K/AKT signaling, the mechanism of PTEN expression regulation in the pathogenesis of CML is incompletely understood.

Lower PTEN expression and activity can result from epigenetic silencing, genomic loss, transcriptional repression, posttranscriptional regulation by microRNAs, and so on (15). Indeed, previous studies showed that miR-21 and miR-181 inhibit PTEN expression by directly targeting its 3′-UTR (16, 17). miR-328, miR-203, and miR-424 play an important role not only in CML development and but also in IM resistance (18–20). miR-188-5p suppresses tumor cell proliferation and metastasis in different types of cancers by targeting FGF5 or ZFP91 (21, 22). However, it remains unknown whether miR-188-5p plays a role in regulating PTEN expression as well as in CML development.

Morin (3,5,7,2′,4′-pentahydroxyflavone), a kind of flavonoid isolated from figs and other Moraceae, was found to exert a potent anticancer activity by inhibiting tumor growth, inducing apoptosis, and reversing the drug resistance in various human tumors, including breast cancer (23), oral cancer (24), prostate cancer (25), multiple myeloma (26), and acute leukemia (27). Moreover, a mechanistic study showed that the anticancer effects of morin are associated with suppression of the AKT signaling pathway (23). However, it remains to be clarified whether anticancer activity of morin against CML involves its regulatory effects on the expression of PTEN and miR-188-5p as well as on AKT activity.

In the present study, we determined whether and how morin exerts an anticancer effect on CML by regulating miR-188-5p and PTEN expression and thus repressing the PI3K/AKT signaling pathway.

Patients and specimens

Thirty patients with CML who were admitted to the Department of Hematology of the Second Hospital of Hebei Medical University from May 2016 to June 2017 were selected as the research objects. Patient characteristics are summarized in Table 1. Thirty healthy donors were selected to serve as controls. Peripheral blood mononuclear cells (PBMC) were isolated by using lymphocyte separation medium according to the manufacturer's instructions. Diagnosis of Ph-positive CML was confirmed by bone marrow morphology, cytogenetic, and molecular biology. And patients did not undergo chemotherapy before the specimens were collected. The study protocol was approved by the Ethics Committee of Second Hospital of Hebei Medical University, and written informed consent was obtained from all the patients.

Table 1.

Patient characteristics

CharacteristicsCML-CP (n = 30)
Age (years), median (range) 42 (29–71) 
Male/female (n/n18/12 
WBCs, × 109/L, median (range) 249 (39–405) 
Hb level (g/L) 99 (81–146) 
PLT count, × 109/L, median (range) 464 (94–797) 
CharacteristicsCML-CP (n = 30)
Age (years), median (range) 42 (29–71) 
Male/female (n/n18/12 
WBCs, × 109/L, median (range) 249 (39–405) 
Hb level (g/L) 99 (81–146) 
PLT count, × 109/L, median (range) 464 (94–797) 

Abbreviations: Hb, hemoglobin; PLT, platelet; WBC, white blood cells.

Cell culture and transfection

Human CML cell lines (KCL22 and K562) and acute myeloid leukemia (AML) cell lines (THP-1 and HL-60) were maintained in the laboratory. KCL22 cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Gibco), with 10% fetal bovine serum (FBS; Clark Bio), 100 units/mL penicillin and 100 μg/mL streptomycin. K562, THP-1, and HL-60 cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS and the two antibiotics listed above. Cell lines were grown at 37°C with 5% CO2. Short tandem repeat analysis was used to tested Mycoplasma contamination.

Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. The miR-188-5p mimic/inhibitor, si-PTEN-1#, si-PTEN-2#, and their respective controls were purchased from GenePharma Co. Ltd.

Cell viability assay

Cell viability was tested by Cell Counting Kit-8 (CCK-8, Beibo) following the manufacturer's protocols. Morin was purchased from Sigma-Aldrich (M4008, Sigma-Aldrich). Briefly, cells were seeded in 96-well plates and treated with morin at the different concentrations (3.375, 6.25, 12.5, 25, 50, 100, 200, 400 μmol/L) for 48 hours, respectively. Finally, the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher USA).

Analyses of apoptosis

Cells grown in 6-well plates were treated with morin at 25 and 50 μmol/L for 48 hours, respectively. For drug combination studies, cells were treated with 25 μmol/L morin and 0.1 μmol/L IM for 48 hours. The Annexin V–FITC/PI apoptosis detection kit (BD Biosciences) was used to detect cell apoptosis following the manufacturer's instructions. The data analysis was performed using BD FACS Diva software (BD).

Xenograft animal model

All animal studies were approved by the Institutional Animal Care Committee of Hebei Medical University. Six-week-old BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. 1 × 107 LV-anti-miR-188-5p or LV-miR-anti-NC–infected K562 cells were resuspended in 50 μL PBS mixed with 50 μL Matrigel (356234; BD); this suspension was injected subcutaneously into the left dorsal flanks. Following a week inoculation period, tumor-bearing mice were randomly divided into the morin treatment and control groups. In the treatment group, morin (50 mg/kg) was administered to mice intraperitoneally five times/week for 2 weeks. And the control group was injected with the saline. The volume of xenograft was measured twice a week. At the end of the experiment, mice were euthanized by carbon dioxide asphyxiation. Tumor tissues were stored in liquid nitrogen or fixed in 4% paraformaldehyde immediately and stored at −80°C until further use.

RNA extraction and quantitative real-time PCR

Total RNA was extracted by using QIAzol Lysis Reagent (79306) according to the manufacturer's protocol. For microRNA analysis, the miScripIIRT kit (QIAGEN GmbH) was used for reverse transcription, and the miScript SYBR Green PCR kit was used for qRT-PCR with specific primers for microRNAs. RNU6b (U6) was used as an internal control. For mRNA analysis, total cellular RNA was reverse-transcribed to first-strand cDNA with M-MLV First-Strand Kit (Life Technologies). And Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used for the qRT-PCR of mRNAs. The mRNA expression was normalized to that of β-actin.

Western blot analysis

The protein was extracted from the cultured cells and frozen tissue samples with RIPA lysis buffer. Equal amounts of protein were run on 10% SDS-PAGE, and electro-transferred to a polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% nonfat milk, the membranes were incubated with specific primary antibodies as follows: anti-PTEN (1:1,000, ab32199), anti-PCNA (1:1,000, ab31163), anti-caspase-3 (1:1,000, ab13847), anti-pan-AKT (1:1,000, ab8805), anti-pan-AKT (phospho T308; 1:1,000, ab38449), anti-BCL-2 (1:1,000, 12,789–1-AP), anti-BAX (1:1,000, 50,599–2-Ig) or anti-β-actin (1:1,000, sc-47,778). The proteins were visualized with Immobilon ECL (Millipore). FusionCapt Advance Fx5 software (Vilber Lourmat) was used to capture the images.

In situ hybridization

In situ hybridization (FISH) was performed following the manufacturer's instructions of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon; ref. 28). In brief, all the cell smears were deparaffinized and rehydrated for fluorescence in situ hybridization. Hybridization was performed using fluorescence-labeled miR-188-5p probes with hybridization buffer (Exiqon) by incubation at 55°C for 1 hours. Then, smears were stringently washed with SSC buffer and PBS. Leica microscope (Leica DM6000B) was used to acquire the images. And all the images were digitized with a software of LAS V.4.4 (Leica).

Vector construction and luciferase reporter assay

Restriction-enzyme digestion and one-step cloning (ClonExpress II One-Step Cloning Kit, C112-02; Vazyme Biotech Co., Ltd.) were used to construct the luciferase reporter plasmids. The 3′-UTR sequences of PTEN containing the miR-188-5p target site (wild-type or mutant) were inserted into pmir-GLO Dual-Luciferase miRNA Target Expression Vector (Promega Corp.) digested by the Xho1 and Sal1. K562 cells were seeded into a 24-well plate, and miR-188-5p mimic (or mimic-NC) was cotransfected with PTEN reporter construct (wild-type or mutant) or the empty vector. Twenty-four hours after transfection, cells were harvested in lysis buffer. The Dual-Glo Luciferase Assay System (Promega) was used to detect luciferase activity according to the manufacturer's protocols. Firefly luciferase (FLuc) activity was measured and normalized against Renilla luciferase (RLuc) activity.

Immunofluorescence staining

Formaldehyde (4%) was used to fix the cells, and cell smears were preincubated with 10% normal goat serum (710,027, KPL). Then, smears were incubated with primary antibody anti-PTEN (22034-1-AP, Proteintech) at 37°C for 1 hour. Secondary antibody was fluoresce-labeled antibody to rabbit IgG (021516, KPL). Finally, smears were incubated with DAPI (157,574, MB Biomedical) for nuclear counterstaining. Images were acquired by using confocal microscopy (DM6000 CFS, Leica) and digitized with LAS AF software.

Target prediction

miRanda (www.microrna.org) and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html) were used to identify the potential target microRNA of PTEN.

Statistical analysis

Data were shown as mean ± standard error of mean. The Student t test was used to detect the significance of differences between two groups. P < 0.05 was considered statistically significant.

Morin inhibits proliferation and induces apoptosis of CML cell lines

Morin (3,5,7,2′,4′-pentahydroxyflavone, C15H10O7) has the structure of one oxygen-containing heterocyclic ring connecting with two aromatic rings (Fig. 1A; ref. 29). Because morin is reported to suppress tumor growth and reduce the viability of breast cancer cells (23), we sought to know whether morin also exerts an anticancer effect on CML cell lines K562 and KCL22. To do this, K562 and KCL22 cells were exposed to the different concentrations of morin for 48 hours, and cell viabilities were tested by using the Cell Counting CCK-8 Kits. The results showed that morin inhibited cell viabilities in a concentration-dependent manner. The IC50 value of morin, a concentration that reduces the cell viability by 50%, was observed at 79.3 μmol/L in K562 cells and 73.04 μmol/L in KCL22 cells (Fig. 1B). Next, K562 and KCL22 cells were treated with morin (25 μmol/L and 50 μmol/L) for 48 hours to determine whether morin could induce CML cell apoptosis. Cell apoptosis was evaluated using flow cytometry-based Annexin V/PI assay. The results showed that morin significantly induced apoptosis in two cell lines. When these two cells were treated with 25 or 50 μmol/L morin, the number of apoptotic cells had a 2- to 3-fold increase, respectively (Fig. 1C). These results were further verified by Western blot analysis, showing that morin obviously decreased proliferating cell nuclear antigen (PCNA) level and dose dependently increased cleaved caspase-3, a hallmark of apoptosis (Fig. 1D). These results indicate that morin significantly inhibits cell growth and induces cell apoptosis in CML cell lines.

Figure 1.

Morin inhibits proliferation and induces apoptosis of CML cell lines. A, The chemical structure of morin. B, K562 and KCL22 cells were treated with morin at different concentrations for 48 hours, respectively. Cell viability was detected by CCK-8 assay. Data shown are mean ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus DMSO control. C, K562 and KCL22 cells were treated with morin (25 μmol/L and 50 μmol/L) for 48 hours. Cell apoptosis was assessed by Annexin V–FITC/PI staining. Right, the apoptosis rate of three independent experiments. ***, P < 0.001 versus DMSO control. D, Cells prepared as in C; Western blot analysis detected the expressions of PCNA protein and cleaved caspase-3 protein. Right, densitometric analysis of three independent experiments. The loading control is β-actin. **, P < 0.01; ***, P < 0.001 versus DMSO control.

Figure 1.

Morin inhibits proliferation and induces apoptosis of CML cell lines. A, The chemical structure of morin. B, K562 and KCL22 cells were treated with morin at different concentrations for 48 hours, respectively. Cell viability was detected by CCK-8 assay. Data shown are mean ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus DMSO control. C, K562 and KCL22 cells were treated with morin (25 μmol/L and 50 μmol/L) for 48 hours. Cell apoptosis was assessed by Annexin V–FITC/PI staining. Right, the apoptosis rate of three independent experiments. ***, P < 0.001 versus DMSO control. D, Cells prepared as in C; Western blot analysis detected the expressions of PCNA protein and cleaved caspase-3 protein. Right, densitometric analysis of three independent experiments. The loading control is β-actin. **, P < 0.01; ***, P < 0.001 versus DMSO control.

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Morin suppresses AKT phosphorylation by upregulating PTEN expression

Activation of PI3K/AKT signaling is involved in the control of cellular proliferation and apoptosis (30), and phosphatase and tensin homologue (PTEN) is a well-known negative regulator of the PI3K/AKT pathway (10). Therefore, we examined the effects of morin on AKT activation and PTEN expression. Because morin played its proapoptotic role through a similar mechanism in the same kind of cell lines (K562 and KCL22), we chose K562 cells to investigate the mechanism underlying proapoptotic effect of morin. K562 cells were treated with morin (25 and 50 μmol/L), and mRNA and protein levels of PTEN were analyzed by qRT-PCR and Western blotting, respectively. We found that morin significantly upregulated PTEN protein expression in a dose-dependent manner but did not significantly affect its mRNA level (Fig. 2A and B). In addition, immunofluorescence staining also confirmed that PTEN protein expression obviously increased in K562 cells treated with morin (Fig. 2C). Then, we determined the effects of morin treatment on the downstream targets of the PTEN/AKT pathways. Compared with the control group, phosphorylated AKT (p-AKT) protein level was dramatically decreased in morin-treated cells. Meanwhile, expression of proapoptotic BAX was significantly upregulated at both protein and mRNA levels, whereas antiapoptotic BCL-2 was significantly decreased (Fig. 2D and E). These results suggest that inhibition of AKT signaling and the proapoptotic action of morin are associated with PTEN upregulation induced by morin.

Figure 2.

Morin suppresses AKT phosphorylation by upregulating PTEN expression. K562 cells were treated with morin (25 and 50 μmol/L) for 48 hours. A and D, Western blot analysis was used to detect PTEN protein level (A) and AKT/BCL-2 downstream protein levels (AKT, p-AKT, BCL-2, and BAX; D). D, Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus DMSO. B and E, qRT-PCR detected PTEN mRNA level (B), and BAX and BCL-2 mRNA levels (E); **, P < 0.01; ***, P < 0.001 versus DMSO. Normalized against β-actin. C, K562 cells were treated with morin (25 μmol/L) for 48 hours. Immunofluorescence staining analysis of PTEN protein level compared with PBMCs of healthy donors. Nuclei were staining with DAPI (blue). Scale bars, 250 μm. F, qRT-PCR was used to detect PTEN mRNA expression in PBMCs of CML patients (n = 30) compared with PBMCs of healthy donors (n = 30). *, P < 0.05 versus PBCMs of healthy donors. G, Western blot analysis was used to detect PTEN protein level in PBMCs of CML patients compared with PBMCs of healthy donors. Blots from a representative experiment are shown on the left, whereas the right shows densitometric analysis of PTEN protein level in PBMCs of 30 CML patients and 30 healthy donors. *, P < 0.05 versus PBCMs of healthy donors. H, qRT-PCR was used to detect the mRNA level of PTEN in CML and AML cell lines (K562, KCL22, THP-1, and HL-60) compared with PBMCs of healthy donors. ***, P < 0.001 versus PBMCs of healthy donors.

Figure 2.

Morin suppresses AKT phosphorylation by upregulating PTEN expression. K562 cells were treated with morin (25 and 50 μmol/L) for 48 hours. A and D, Western blot analysis was used to detect PTEN protein level (A) and AKT/BCL-2 downstream protein levels (AKT, p-AKT, BCL-2, and BAX; D). D, Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus DMSO. B and E, qRT-PCR detected PTEN mRNA level (B), and BAX and BCL-2 mRNA levels (E); **, P < 0.01; ***, P < 0.001 versus DMSO. Normalized against β-actin. C, K562 cells were treated with morin (25 μmol/L) for 48 hours. Immunofluorescence staining analysis of PTEN protein level compared with PBMCs of healthy donors. Nuclei were staining with DAPI (blue). Scale bars, 250 μm. F, qRT-PCR was used to detect PTEN mRNA expression in PBMCs of CML patients (n = 30) compared with PBMCs of healthy donors (n = 30). *, P < 0.05 versus PBCMs of healthy donors. G, Western blot analysis was used to detect PTEN protein level in PBMCs of CML patients compared with PBMCs of healthy donors. Blots from a representative experiment are shown on the left, whereas the right shows densitometric analysis of PTEN protein level in PBMCs of 30 CML patients and 30 healthy donors. *, P < 0.05 versus PBCMs of healthy donors. H, qRT-PCR was used to detect the mRNA level of PTEN in CML and AML cell lines (K562, KCL22, THP-1, and HL-60) compared with PBMCs of healthy donors. ***, P < 0.001 versus PBMCs of healthy donors.

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To investigate the clinical significance of PTEN expression, we examined PTEN protein and mRNA expression in patients (n = 30) with newly diagnosed CML-CP. As shown in Fig. 2F, PTEN mRNA level was significantly downregulated in CML patients compared with that of healthy donors (n = 30). Consistently, Western blot analysis also showed that PTEN protein was markedly decreased in CML patients (Fig. 2G). Further, we examined PTEN expression in the different CML cell lines (KCL22 and K562) and AML cell lines (HL-60 and THP-1) as well as in normal human PBMCs and showed that PTEN mRNA and protein expressions were significantly decreased in CML and AML cell lines compared with those in normal human PBMCs (Fig. 2H). Because a previous study confirmed that IM resistance in patients with ALL was associated with PTEN downregulation (31), and we found that morin induced the apoptosis of CML cells by upregulating PTEN expression, we investigated whether morin could enhance the sensitivity of CML cells to IM. K562 cells were treated with morin (25 μmol/L) and different concentrations of IM (0–1 μmol/L) for 48 hours. As shown in Supplementary Fig. S1, IM combined with 25 μmol/L morin markedly reduced K562 cell viability in an IM dose-dependent manner compared with that treated with IM alone. IC50 of IM was significantly decreased in the presence of 25 μmol/L morin. Correspondingly, morin combination with IM led to a significant increase in the apoptosis of K562 cells compared with that treated with IM alone (Supplementary Fig. S2).These findings suggest that PTEN downregulation may be responsible for the development of human CML.

PTEN plays an essential role in morin-induced apoptosis of CML cells

The above results suggest that morin induced apoptosis of CML cells and upregulated PTEN expression; we next investigated the mutual relationship between morin-induced apoptosis of CML cells and PTEN expression level. First, we synthesized two small interfering RNAs against PTEN (si-PTEN-1# and si-PTEN-2#) and confirmed that transfecting K562 cells with these two si-RNAs could significantly downregulate PTEN mRNA and protein expression relative to si-NC (Fig. 3A and B). Comparatively, si-PTEN-2# had higher knockdown efficiency than that of si-PTEN-1#. So si-PTEN-2# was chosen for all subsequent experiments. To demonstrate the involvement of PTEN in morin-induced K562 cell apoptosis, K562 cells were transfected with si-PTEN, followed by treatment with morin. Western blot analysis showed that knockdown of PTEN by its siRNA completely abrogated morin-induced increase in PTEN expression and caspase-3 cleavage. Simultaneously, morin-suppressed cell proliferation was rescued by PTEN knockdown, as evidenced by PCNA expression changes (Fig. 3C). We also determined the effects of PTEN knockdown on AKT phosphorylation and the expression of BCL-2 and BAX. The results indicated that PTEN knockdown abolished the inhibitory effect of morin on AKT phosphorylation and thus attenuated the upregulation of BAX induced by morin (Fig. 3D). Consistently, flow-cytometric analysis showed that knockdown of PTEN by its siRNA significantly reduced the cell apoptosis induced by morin (Fig. 3E). Furthermore, knockdown of PTEN in combination with morin treatment significantly increased IC50 values of morin in K562 cells compared with morin alone (Fig. 3F). These findings indicate that PTEN plays an essential role in morin-induced apoptosis of K562 cells.

Figure 3.

PTEN plays an essential role in morin-induced apoptosis of CML cells. A and B, K562 cells were transfected with si-PTEN-1#, si-PTEN-2#, or si-NC; qRT-PCR (A) and Western blot analysis (B) detected the PTEN mRNA and protein level, respectively. Right, densitometric analysis of three independent experiments. ***, P < 0.001 versus si-NC control. C and D, K562 cells were transfected with si-PTEN or si-NC and then treated with or without morin (25 μmol/L) for 48 hours. PTEN, PCNA, cleaved caspase-3, and AKT pathway downstream protein levels (AKT, p-AKT, BCL-2, and BAX) were detected by Western blotting. Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus their corresponding control. E, K562 cells were treated as described in C; cell apoptosis was assessed by Annexin V–FITC/PI staining. Bottom, the apoptosis rate of three independent experiments. **, P < 0.01 versus corresponding control. F, K562 cells were transfected with si-PTEN or si-NC and treated with morin at different concentrations for 48 hours. IC50 of morin was detected by the CCK-8 assay. ***, P < 0.001 versus si-NC control.

Figure 3.

PTEN plays an essential role in morin-induced apoptosis of CML cells. A and B, K562 cells were transfected with si-PTEN-1#, si-PTEN-2#, or si-NC; qRT-PCR (A) and Western blot analysis (B) detected the PTEN mRNA and protein level, respectively. Right, densitometric analysis of three independent experiments. ***, P < 0.001 versus si-NC control. C and D, K562 cells were transfected with si-PTEN or si-NC and then treated with or without morin (25 μmol/L) for 48 hours. PTEN, PCNA, cleaved caspase-3, and AKT pathway downstream protein levels (AKT, p-AKT, BCL-2, and BAX) were detected by Western blotting. Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus their corresponding control. E, K562 cells were treated as described in C; cell apoptosis was assessed by Annexin V–FITC/PI staining. Bottom, the apoptosis rate of three independent experiments. **, P < 0.01 versus corresponding control. F, K562 cells were transfected with si-PTEN or si-NC and treated with morin at different concentrations for 48 hours. IC50 of morin was detected by the CCK-8 assay. ***, P < 0.001 versus si-NC control.

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miR-188-5p upregulation in CML cells represses PTEN expression by directly targeting its 3′-UTR

The above findings raise an important question about how PTEN expression is downregulated in CML cells. Because it is well known that the expression of many genes is regulated by miRNAs at the posttranscriptional level, we identified potential miRNAs targeting 3′-UTR of PTEN by using two target prediction programs, miRanda and TargetScan, and found 10 putative PTEN-targeting miRNAs. Among them, only miR-188-5p expression was significantly downregulated by morin in K562 cells (Fig. 4A and B). RNA in situ hybridization also confirmed that morin treatment obviously decreased miR-188-5p expression (Fig. 4C).

Figure 4.

miR-188-5p represses PTEN expression by directly targeting the 3′-UTR of PTEN. A, K562 cells were treated with morin (25 μmol/L) for 48 hours. Expressions of 10 potential miRNAs regulating PTEN were determined by qRT-PCR. Normalized against an internal control U6 RNA. *, P < 0.05 versus DMSO control. B, qRT-PCR was used to detect the miR-188-5p expression of K562 cells that were treated with morin (25 and 50 μmol/L) for 48 hours. *, P < 0.05; ***, P < 0.001 versus DMSO control. C, K562 cells were treated with morin (25 μmol/L) for 48 hours and fluorescence in situ hybridization (FISH) was used to detect the miR-188-5p expression. Blue staining represents the nucleus and red staining indicates miR-188-5p. Scale bar, 64 μm. D, Prediction of the miR-188a-5p binding site at PTEN 3′-UTR. Red color indicates the sequence of the mutated miR-188-5p binding site. E, K562 cells were transfected with miR-188-5p mimic, mimic-NC, miR-188-5p inhibitor, or inhibitor-NC, respectively. miR-188-5p expression was detected by qRT-PCR. ***, P < 0.001 versus their corresponding control. F, Luciferase reporter assays were performed in K562 cells cotransfected cells with miR-188-5p mimic and WT or mutant (mut) PTEN 3′-UTR-luciferase reporter; **, P < 0.01 versus mimic-NC. G, K562 cells were prepared as (E); PTEN, AKT, and p-AKT expression was analyzed by Western blotting. Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus their corresponding control. H, qRT-PCR detected the expression of miR-188-5p expression in PBMCs of CML patients compared with PBMCs of healthy donors. **, P < 0.01 versus PBCMs of healthy donors. I, FISH detected miR-188-5p in PBMCs of CML patients and of healthy donors. Blue staining represents the nucleus and red staining indicates miR-188-5p. Scale bar, 64 μm. J, qRT-PCR detected the expression of miR-188a-5p in different CML and AML cell lines (K562, KCL22, THP-1, and HL-60) compared with PBMCs of healthy donors; ***, P < 0.001 versus PBMCs of healthy donors.

Figure 4.

miR-188-5p represses PTEN expression by directly targeting the 3′-UTR of PTEN. A, K562 cells were treated with morin (25 μmol/L) for 48 hours. Expressions of 10 potential miRNAs regulating PTEN were determined by qRT-PCR. Normalized against an internal control U6 RNA. *, P < 0.05 versus DMSO control. B, qRT-PCR was used to detect the miR-188-5p expression of K562 cells that were treated with morin (25 and 50 μmol/L) for 48 hours. *, P < 0.05; ***, P < 0.001 versus DMSO control. C, K562 cells were treated with morin (25 μmol/L) for 48 hours and fluorescence in situ hybridization (FISH) was used to detect the miR-188-5p expression. Blue staining represents the nucleus and red staining indicates miR-188-5p. Scale bar, 64 μm. D, Prediction of the miR-188a-5p binding site at PTEN 3′-UTR. Red color indicates the sequence of the mutated miR-188-5p binding site. E, K562 cells were transfected with miR-188-5p mimic, mimic-NC, miR-188-5p inhibitor, or inhibitor-NC, respectively. miR-188-5p expression was detected by qRT-PCR. ***, P < 0.001 versus their corresponding control. F, Luciferase reporter assays were performed in K562 cells cotransfected cells with miR-188-5p mimic and WT or mutant (mut) PTEN 3′-UTR-luciferase reporter; **, P < 0.01 versus mimic-NC. G, K562 cells were prepared as (E); PTEN, AKT, and p-AKT expression was analyzed by Western blotting. Bottom, densitometric analysis. **, P < 0.01; ***, P < 0.001 versus their corresponding control. H, qRT-PCR detected the expression of miR-188-5p expression in PBMCs of CML patients compared with PBMCs of healthy donors. **, P < 0.01 versus PBCMs of healthy donors. I, FISH detected miR-188-5p in PBMCs of CML patients and of healthy donors. Blue staining represents the nucleus and red staining indicates miR-188-5p. Scale bar, 64 μm. J, qRT-PCR detected the expression of miR-188a-5p in different CML and AML cell lines (K562, KCL22, THP-1, and HL-60) compared with PBMCs of healthy donors; ***, P < 0.001 versus PBMCs of healthy donors.

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Next, computer-based sequence analysis (TargetScan and miRanda) was used to search for the potential matching site of miR-188-5p in PTEN 3′-UTR. As shown in Fig. 4D, there exists a putative miR-188-5p binding site in the PTEN 3′-UTR (Fig. 4D). Then, K562 cells were transfected with miR-188-5p mimic or its inhibitor, and transfection efficiency was detected by qRT-PCR. The results showed that miR-188-5p mimic dramatically increased, whereas its inhibitor markedly decreased the miR-188-5p expression (Fig. 4E). Next, K562 cells were cotransfected with miR-188-5p mimic and PTEN 3′-UTR-luciferase reporter containing wild-type (WT) or mutated (mut) miR-188-5p-binding site. The dual-luciferase reporter assay confirmed that transfecting K562 cells with miR-188-5p mimic significantly decreased the luciferase activity driven by WT PTEN 3′-UTR. However, mutation of the miR-188-5p-binding site in PTEN 3′-UTR abolished the inhibitory effect of miR-188-5p on luciferase activity (Fig. 4F). Further, we transfected miR-188-5p mimic or its inhibitor into K562 cells and detected PTEN and phosphorylated AKT (p-AKT) by using Western blot analysis. The results showed that transfection of miR-188-5p mimic significantly reduced the PTEN level and increased the p-AKT level, while silencing of miR-188-5p by its specific inhibitor increased PTEN protein level and reduced AKT phosphorylation in K562 cells (Fig. 4G). These findings clearly indicate that miR-188-5p negatively regulates PTEN expression by directly targeting its 3′-UTR.

To further identify whether the expression level of miR-188-5p was changed in CML patients, we detected its expression in PBMCs of 30 patients with CML and 30 healthy donors. The qRT-PCR revealed that miR-188-5p expression level was significantly higher in CML PBMCs than in PBMCs of healthy donors (Fig. 4H). Further, RNA in situ hybridization also showed that miR-188-5p expression was markedly upregulated in PBMCs of CML patients compared with that in healthy donors (Fig. 4I). In further experiments, miR-188-5p expression was examined in the different CML cell lines (KCL22 and K562) and AML cell lines (HL-60 and THP-1) as well as in healthy PBMCs, respectively. As expected, the miR-188-5p level was significantly increased in CML cell lines, but not AML cell lines compared with the healthy human PBMCs, with the miR-188-5p level being about 1.5-fold upregulated in CML cell lines (Fig. 4J). Collectively, these results strongly indicated that miR-188-5p upregulation-repressed expression of PTEN is closely associated with CML development.

miR-188-5p downregulation induced by morin enhances CML cell apoptosis by relieving miR-188-5p repression of PTEN expression

In the present study, morin induced CML cell apoptosis and repressed miR-188-5p expression. To clarify the intrinsic relationship between morin-induced miR-188-5p downregulation and cell apoptosis, we investigated the effects of miR-188-5p knockdown on cell apoptosis. As shown in Fig. 5A, depletion of miR-188-5p by a miR-188-5p inhibitor resulted in a significant increase of PTEN expression, accompanied by a significant induction of cell apoptosis, as evidenced by the increased caspase-3 cleavage and BAX expression, as well as by the decreased expression of BCL-2 and PCNA. Correspondingly, the number of apoptotic cells was markedly increased in miR-188-5p-depleted K562 cells compared with that transfected with inhibitor-NC (Fig. 5B). These findings indicate that the downregulation of miR-188-5p facilitates CML cell apoptosis.

Figure 5.

miR-188-5p downregulation induced by morin enhances CML cell apoptosis by relieving miR-188-5p repression of PTEN expression. A, K562 cells were transfected with miR-188-5p inhibitor or inhibitor-NC. Western blot detected PTEN, PCNA, and cleaved caspase-3, BCL-2, BAX protein expressions. Right, densitometric analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus si-NC control. B, K562 cells were treated as described in A; cell apoptosis was assessed by Annexin V–FITC/PI staining. Right, apoptosis rate. **, P < 0.01 ; ***, P < 0.001 versus inhibitor-NC. C, K562 cells were transfected with miR-188-5p mimic or mimic-NC and then treated with or without morin (25 μmol/L) for 48 hours. PTEN, PCNA, and cleaved caspase-3 protein levels were detected by Western blotting. Bottom left, densitometric analysis. *, P < 0.05; **, P < 0.01 versus corresponding control. D, K562 cells were treated as described in C; cell apoptosis was assessed by Annexin V–FITC/PI staining. Bottom left, the apoptosis rate. **, P < 0.01; ***, P < 0.001 versus corresponding control.

Figure 5.

miR-188-5p downregulation induced by morin enhances CML cell apoptosis by relieving miR-188-5p repression of PTEN expression. A, K562 cells were transfected with miR-188-5p inhibitor or inhibitor-NC. Western blot detected PTEN, PCNA, and cleaved caspase-3, BCL-2, BAX protein expressions. Right, densitometric analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus si-NC control. B, K562 cells were treated as described in A; cell apoptosis was assessed by Annexin V–FITC/PI staining. Right, apoptosis rate. **, P < 0.01 ; ***, P < 0.001 versus inhibitor-NC. C, K562 cells were transfected with miR-188-5p mimic or mimic-NC and then treated with or without morin (25 μmol/L) for 48 hours. PTEN, PCNA, and cleaved caspase-3 protein levels were detected by Western blotting. Bottom left, densitometric analysis. *, P < 0.05; **, P < 0.01 versus corresponding control. D, K562 cells were treated as described in C; cell apoptosis was assessed by Annexin V–FITC/PI staining. Bottom left, the apoptosis rate. **, P < 0.01; ***, P < 0.001 versus corresponding control.

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To provide additional confirmation that miR-188-5p downregulation induced by morin was involved in CML cell apoptosis. K562 cells were transfected with miR-188-5p mimic or mimic-NC, followed by treatment with morin or not. Western blot analysis showed that morin-induced upregulation of PTEN was abrogated by miR-188-5p mimic transfection (Fig. 5C). Consistently, morin treatment combined with miR-188-5p overexpression also reduced cell apoptosis induced by morin, as evidenced by the decreased caspase-3 cleavage (Fig. 5C) as well as by flow-cytometric analysis (Fig. 5D). In short, all the outcomes above indicate that miR-188-5p downregulation induced by morin facilitates the apoptosis of CML cells via relieving miR-188-5p repression of PTEN expression.

To further clarify whether morin enhanced chemosensitivity of CML cells to IM by downregulating miR-188-5p, K562 cells were transfected with miR-188-5p mimic or mimic-NC and then incubated with the different concentrations of IM in the presence or absence of 25 μmol/L morin. The results showed that overexpression of miR-188-5p abrogated the inhibitory effect of morin on cell growth, and IC50 of IM was also increased accordingly (Supplementary Fig. S3). Consistent with the results of cell viability assay, overexpression of miR-188-5p in morin-treated cells significantly attenuated cell apoptosis induced by morin (Supplementary Fig. S4). Collectively, these observations strongly support the hypothesis that morin, as an adjuvant, enhances K562 cell sensitivity to IM by downregulating miR-188-5p expression.

Morin reduces CML xenograft growth in vivo

Next, we wanted to further confirm the above findings that morin reduced CML cell growth through the miR-188-5p–regulated PTEN pathway by using a nude mouse xenograft model. First, we established K562 cells stably expressing anti-miR-188-5p by infecting cells with LV-anti-miR-188-5p. Next, nude mice were subcutaneously implanted with the above-established cells and then treated or untreated with morin. As shown in Fig. 6A and B, morin or anti-miR-188-5p alone significantly reduced tumor growth compared with the negative control. As expected, morin treatment combined with LV-anti-miR-188-5p infection further attenuated the volume of xenograft tumors. Similarly, compared with anti-miR-NC–infected mice, the weight of xenograft tumors was significantly decreased in morin-treated mice or miR-188-5p–depleted mice. The weight of tumors was much lower in miR-188-5p–depleted together with morin-treated mice than in morin-treated mice alone (Fig. 6C). In further experiments, the expression of PTEN, PCNA, AKT, p-AKT, and cleaved caspase-3 was detected in xenograft tumors by Western blot analysis. As shown in Fig. 6D, the levels of PTEN and cleaved caspase-3 were dramatically increased in morin-treated or miR-188-5p–depleted mice, especially in their combination, while the expression of PCNA and p-AKT was obviously decreased in morin-treated or miR-188-5p–depleted mice. In addition, morin also significantly suppressed miR-188-5p expression in xenograft tumors (Fig. 6E). These data again suggest that morin reduces CML cell growth in vivo by inhibiting miR-188-5p expression and thus relieving miR-188-5p repression of PTEN expression.

Figure 6.

Morin reduces CML xenograft growth in vivo. K562 cells engineered to stably express anti-miR-188-5p (LV-anti-miR-188-5p) or negative control (LV-miR-NC) were injected subcutaneously in 200 μL 1640/Matrigel (100:100) into the right posterior ankle of the nude mice to establish xenograft tumors. From the eighth day, mice were intraperitoneally injected with morin (50 mg/kg) five times per week. A, Tumor volumes were monitored by direct measurement with calipers and calculated by the formula: (length × width2)/2. **, P < 0.01 versus LV-miR-NC; ##, P < 0.01 versus LV-anti-miR-NC + morin (each group, n = 8). B, Images of tumors excised from each group of mice. C, Average weight of tumors in nude mice. *, P < 0.05; **, P < 0.01 versus corresponding control. D, Total proteins were extracted from excised tumors, and the levels of PTEN, PCNA, AKT, p-AKT, and cleaved caspase-3 were determined by Western blot analysis. E, RNA was extracted from excised tumors and the expression of miR-188-5p was determined by qRT-PCR. **, P < 0.01; ***, P < 0.001 versus corresponding control.

Figure 6.

Morin reduces CML xenograft growth in vivo. K562 cells engineered to stably express anti-miR-188-5p (LV-anti-miR-188-5p) or negative control (LV-miR-NC) were injected subcutaneously in 200 μL 1640/Matrigel (100:100) into the right posterior ankle of the nude mice to establish xenograft tumors. From the eighth day, mice were intraperitoneally injected with morin (50 mg/kg) five times per week. A, Tumor volumes were monitored by direct measurement with calipers and calculated by the formula: (length × width2)/2. **, P < 0.01 versus LV-miR-NC; ##, P < 0.01 versus LV-anti-miR-NC + morin (each group, n = 8). B, Images of tumors excised from each group of mice. C, Average weight of tumors in nude mice. *, P < 0.05; **, P < 0.01 versus corresponding control. D, Total proteins were extracted from excised tumors, and the levels of PTEN, PCNA, AKT, p-AKT, and cleaved caspase-3 were determined by Western blot analysis. E, RNA was extracted from excised tumors and the expression of miR-188-5p was determined by qRT-PCR. **, P < 0.01; ***, P < 0.001 versus corresponding control.

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CML was the first neoplasm associated with a chromosomal aberration, known as the Philadelphia chromosome, and was characterized by a constitutively active BCR–ABL tyrosine kinas. Although CML patients receiving IM mesylate (IM), a BCR–ABL1 tyrosine kinase inhibitor (TKI), could transform chronic-phase CML from a universally fatal disease to a chronic illness (32, 33), not all CML patients benefit greatly or equally from IM due to drug intolerance and drug resistance (34–36). Thus, it is necessary to develop additional therapeutic drugs to overcome IM intolerance and resistance in CML patients. Morin, originally isolated from members of the Moraceae family, has been shown to inhibit growth and induce apoptosis in various cancer cells (23, 26, 37). Previous studies demonstrated that morin has a very low toxicity and is well tolerated (38). In addition, morin might exert protective effects against oxidative stress and inflammation and thus could reduce negative side effects of several drugs, without interfering with their functions (25, 39). All these findings suggest that morin could be used, either alone or in combination with other drugs, to treat many human cancers.

BCR–ABL tyrosine kinase activates multiple signal transduction pathways, including MAPK/ERK, PI3K/AKT, and STAT3/STAT5, all of which are responsible for cell proliferation and survival (7). Morin exerts its anticancer activity through inhibiting AKT signaling. For example, it has been demonstrated that morin reduced breast cancer MDA-MB-231 cell growth and invasion by decreasing the phosphorylation of AKT (23). Similarly, morin also induced apoptosis of HL-60 cells by reduction of antiapoptotic BCL-2 expression, which is negatively regulated by AKT (27). In addition, morin prevented the growth and dissemination of metastatic cancer cells by inhibiting the activity of NF-kB, a downstream protein of the AKT pathway (40). However, the underlying mechanisms of the apoptosis induced by morin in CML cells are still poorly understood. In this study, we confirmed that morin inhibited proliferation and induced apoptosis of CML cells in vivo and in vitro. As expected, inhibition of AKT signaling by morin and proapoptotic action of morin were associated with the upregulation of PTEN induced by morin. More importantly, we found for the first time that morin upregulated PTEN protein expression in CML cells. Furthermore, morin reduced AKT phosphorylation and BCL-2 expression, but increased BAX expression. It can be concluded that anticancer activity of morin against CML is due to PTEN upregulation and inhibition of AKT signaling induced by morin, which in turn regulated BCL-2/Bax expression.

PTEN, acting as a negative regulator of the PI3K/AKT pathway, plays a key role in cell biological functions, including proliferation, apoptosis, metabolism, and cell survival (41). It is well known that, as a tumor suppressor, PTEN is downregulated or mutated in multiple types of cancer, such as lung cancer (42), breast cancer (43), and colorectal carcinoma (44). Furthermore, mutations and deletions of PTEN were found in some human hematologic malignancies. For example, Zhang reported that mice with PTEN deletion in hematopoietic stem cells were susceptible to the myeloid and T-lymphoid lineage disorder. Moreover, mice engrafted with PTEN-deficient HSCs inhibited the development of hematopoietic cells and increased the occurrence of AML; ref. 45). Palomero showed that NOTCH1 regulated PTEN expression and AKT signaling, and the mutation of NOTCH1 resulted in the loss of PTEN and hyperactivation of the PI3K/AKT signaling in T-cell lymphoblastic leukemias (T-ALL; ref. 46). Specific to CML, PTEN activity and expression were negatively regulated by BCR–ABL1. However, overexpression of PTEN inhibited the development of CML and B-ALL induced by BCR–ABL (47). In the present study, we found that PTEN was downregulated in CML patients and CML cell lines, and upregulation of PTEN was closely associated with CML cell apoptosis. We further confirmed that morin treatment significantly increased PTEN protein level, whereas its mRNA level was not altered. Therefore, morin may be a posttranscriptional regulator of PTEN.

miRNAs play a critical role in tumor progress by modulating gene expression at the posttranscriptional level. Over the past decade, accumulating evidence shows that PTEN expression is regulated by several miRNAs at the posttranscriptional level, and abnormal expression of miRNAs is responsible for the development of hematologic malignancies. For example, PTEN has been identified as a target gene of miR-21 in MDS and associated with TGFβ signaling activation (48). miR-205-5p was upregulated in MDS cells and negatively regulated PTEN expression by directly targeting PTEN 3′-UTR (49). Overexpression of miR-17/92 has been found in lymphoid malignancies, and upregulation of miR-17/92 was closely correlated with expression of the proapoptotic factor Bim (50). miR-3142 activated PI3K/AKT signaling by reducing PTEN expression in CML cells (51). Notably, a previous study showed that miR-188-5p could directly target PTEN 3′-UTR and posttranscriptionally regulated PTEN expression in diabetic kidney disease (52). More importantly, we found that miR-188-5p was upregulated in CML cells, and increased miR-188-5p repressed PTEN expression by directly targeting its 3′-UTR. Morin upregulated PTEN expression and thus attenuated AKT signaling through suppressing miR-188-5p expression in CML cells. Downregulation of miR-188-5p expression induced by morin in K562 cells inhibited cell viability and promoted apoptosis. The xenograft model of CML with K562 cells stably expressing anti-miR-188-5p also exhibited that depletion of miR-188-5p in CML cells could inhibit the tumor growth. It is possible that miR-188-5p might also target additional target genes, as a single miRNA can control a set of target genes related to CML development. Therefore, further investigations are required to fully understand the role of miR-188-5p in CML development.

In conclusion, morin, as a PTEN/AKT inhibitor, induces apoptosis of CML cells through suppressing miR-188-5p expression. Hence, morin may be a promising adjuvant to treat patients with CML.

No potential conflicts of interest were disclosed.

The present study was authorized by the Ethics Committee of Second Hospital of Hebei Medical University, and verbal consent was obtained from each patient. All patients and volunteers were anonymous and provided written informed consent.

All animal studies were approved by the Institutional Animal Care and Use Committee of Hebei Medical University approval (ID: HebMU 20080026), and all efforts were made to minimize suffering.

Conception and design: Z.-Y. Nie, L. Yang, J.-M. Luo

Development of methodology: Z.-Y. Nie, X.-J. Liu, Z. Yang, G.-S. Yang, J. Yu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z.-Y. Nie, Z. Yang, G.-S. Yang, J. Zhou, Y. Qin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Yang

Writing, review, and/or revision of the manuscript: Z.-Y. Nie, L. Yang, L.-L. Jiang, J.-M. Luo

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.-M. Luo

Study supervision: J.-K. Wen, J.-M. Luo

The authors thank Yin-Tao Shang and Jing-Ci Yang for technical assistance. J.M. Luo was supported by the Natural Science Foundation of Hebei Province of China (no. H2018206035).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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